, 2009; Lai et al., 2005; Leenders et al., 2008). Olaparib Accordingly, we conducted cell-surface biotinylation assays to examine whether Cdk5/p35 increases CaV2.2 surface expression in our heterologous system. However, CaV2.2 surface expression was not upregulated by Cdk5/p35 in stable cell lines or after coexpression with WT CaV2.2 or 8X CaV2.2, indicating that altered channel surface expression was not responsible for the enhancement of the CaV2.2 current density by Cdk5/p35-mediated phosphorylation (Figures 4A and 4B; Table S3). We next assessed channel open probability (Po) of CaV2.2 in the presence of Cdk5/p35 as previously
described (Agler et al., 2005). To obtain maximal channel open probability, Po, for each cell, the maximal ionic current conductance, Gmax (Figure 4C), was plotted as a function of the integral of the channel gating current at the reversal potential Qmax (Figure 4D). Interestingly, Cdk5/p35-mediated phosphorylation
of CaV2.2 increased the channel open probability, LBH589 Po (Figures 4E and 4F). Importantly, the Cdk5/p35-mediated increase in the WT CaV2.2 channel open probability was not observed in 8X CaV2.2. To further determine whether the dramatic increase in CaV2.2 current density impacts CaV2.2 surface expression in primary neurons, we cloned the full-length WT CaV2.2 α1 subunit or the 8X CaV2.2 α1 subunit cDNA into a bicistronic herpes simplex virus (HSV) backbone that coexpresses green fluorescent protein (GFP) (Neve et al., 2005). In primary neurons, transduction with HSV yields about 90% GFP-positive cells after 24 hr (Figure S4A). Upon transduction of primary neurons with WT CaV2.2 or 8X CaV2.2 HSV, however, there were no alterations in CaV2.2 surface levels compared to neurons transduced 17-DMAG (Alvespimycin) HCl with control GFP HSV (Figure S4B). Collectively, these data suggest that in addition to increased channel availability, Cdk5-mediated phosphorylation of CaV2.2 results in increased calcium influx due to enhanced channel open probability. We predicted that Cdk5-mediated phosphorylation of CaV2.2 might play an important physiological role in CaV2.2-mediated neurotransmission. To test this hypothesis,
whole-cell CaV2.2 currents were isolated in neurons transduced with HSV expressing control GFP, WT CaV2.2, or 8X CaV2.2. Consistent with our heterologous cell data, Cdk5-mediated phosphorylation of WT CaV2.2, but not 8X CaV2.2, increased neuronal CaV2.2 current density when compared to neurons expressing control GFP HSV (Figure 5A; Table S4). Furthermore, inhibition of Cdk5 activity using a dominant-negative Cdk5 (DNK5) HSV further reduced CaV2.2 current density (Figure 5A), suggesting that Cdk5 is the major kinase responsible for CaV2.2 phosphorylation and increased CaV2.2 current density. We also examined whether the P/Q-type calcium channel (CaV2.1), the other major presynaptic calcium channel in neurons, was affected by expression of WT CaV2.2 or 8X CaV2.2 HSV but found no differences in CaV2.